We identified nine nucleotide differences between the genomes of dengue-2 (DEN-2) 16681 virus and its vaccine derivative, strain PDK-53. These included a C-to-T (16681-to-PDK-53) mutation at nucleotide position 57 of the 5'-untranslated region, three silent mutations, and substitutions prM-29 Asp to Val, NS1-53 Gly to Asp, NS2A-181 Leu to Phe, NS3-250 Glu to Val, and NS4A-75 Gly to Ala. Unpassaged PDK-53 vaccine contained two genetic variants as a result of partial mutation at NS3-250. We constructed infectious cDNA clones for 16681 virus and each of the two PDK-53 variants. DEN-2 16681 clone-derived viruses were identical to the 16681 virus in plaque size and replication in LLC-MK2 cells, replication in C6/36 cells, E and prM epitopes, and neurovirulence for suckling mice. PDK-53 virus and both clone-derived PDK-53 variants were attenuated in mice. However, the variant containing NS3-250-Glu was less temperature sensitive and replicated better in C6/36 cells than did PDK-53 virus. The variant containing NS3-250-Val had smaller, more diffuse plaques, decreased replication, and increased temperature sensitivity in LLC-MK2 cells relative to PDK-53 virus. Both PDK-53 virus and the NS3-250-Val variant replicated poorly in C6/36 cells relative to 16681 virus. Unpassaged PDK-53 vaccine virus and the virus passaged once in LLC-MK2 cells had genomes of identical sequence, including the mixed NS3-250-Glu/Val locus. Although the NS3-250-Val mutation clearly affected virus replication in vitro, it was not a major determinant of attenuation for PDK-53 virus in suckling mice.